The formation of synaptic connections is the grand finale in the development of the nervous system. When the growth cone of an axon comes into contact with its synaptic target, a series of changes takes place that transforms it from a sensor for pathway seeking to a highly efficient nerve terminal capable of activity-regulated secretion of neurotransmitters. The nerve terminal, characterized by the highly organized clusters of synaptic vesicles at the site of transmitter release and the elaborate molecular complex for the activity- dependent exocytosis of these vesicles at the presynaptic membrane, is a remarkable example of cellular differentiation. Recent studies have suggested that the extracellular matrix surrounding the target cell harbors molecular cues that instruct the growth cone to undergo presynaptic differentiation. This study is aimed at understanding the molecular interactions between the pre- and postsynaptic cells that lead to the formation of the nerve terminal. The development of the nerve terminal at the neuromuscular junction will be used as the main model for presynaptic differentiation of this study. The aims of this study are: (1) to study the role of peptide growth factors that are bound to the extracellular matrix of the skeletal muscle in signaling presynaptic development; (2) to understand the role of the cytoskeleton in the assembly of synaptic vesicle clusters; (3) to characterize the movement and clustering of synaptic vesicles within the neuronal process during presynaptic development. Tissue cultures of spinal cord neurons from the amphibian Xenopus embryos and hippocampal neurons from normal mice and mice with deficiency in the synaptic-vesicle protein synapsin will be used in this study. The presynaptic differentiation will be induced by coculturing neurons with muscle cells or with other neurons, or by applying growth factor-coated microbeads to neurons to mimic the target contact. A range of techniques, including light and electron microscopy, immunochemistry, protein biochemistry, molecular biology and electrophysiology, will be used to observe the development of the nerve terminal in response to synaptogenic signals. The overall goal is to understand the signal transduction mechanism involved in presynaptic differentiation at the neuromuscular junction as well as within the central nervous system. Many neurological and neuromuscular disorders are results of malfunction of synaptic connections in the nervous system. The principles garnered from this study should lead to a better understanding of the causes of these maladies.